BIO461 MICROBIOLOGY

MICROBIOLOGY
PRACTICAL BOOKLET
BIO 461

BSc(Hons.) Biology - BIO461

Ernie Eileen Rizlan Ross

BIO461 MICROBIOLOGY

DEPARTMENT OF BIOLOGICAL SCIENCES

FACULTY OF APPLIED SCIENCES
UNIVERSITI TEKNOLOGI MARA

MICROBIOLOGY PRACTICAL BOOKLET

BIO 461

COMPILED AND EDITED BY

ERNIE EILEEN RIZLAN ROSS
2022
BIO461 MICROBIOLOGY

INTRODUCTORY MICROBIOLOGY

The first year Microbiology Practical Booklet contains a variety of experiments designed to
introduce students to the safe handling of micro-organisms, the use of the microscope, the
assessment of growth of micro-organisms and the characterization and identification of
bacteria and fungi. In carrying out these tasks students are expected to adopt a mature attitude
towards the subject because of the potential health risk involved in microbiological work. All
micro-organisms have the potential to cause disease and students will be expected to adhere
strictly to the safety regulations governing the use of the Microbiology Laboratory.

ASSESSMENT OF PRACTICAL WORK

During your practical work, you will be expected to make notes, diagrams etc and in order to
test your ability to present scientific reports, designated practicals must be fully written up,
with the Title, Objective(s), Introduction, Materials and Methods, Results and Discussion
sections. The criteria on which the grading will depend on will be :

i. Presentation of reports to a deadline

ii. Accuracy of results
iii. Overall quality of presentation
iv. Quality of data
v. Quality of discussion

GENERAL LABORATORY INSTRUCTIONS

1. A clean, white lab coat must be worn in the microbiology laboratory.

Remember No Lab Coat, No Practical!

2. Hands should be washed on entering and must be washed before leaving the lab.

3. Disinfect the lab tables before and at the conclusion of each lab.

4. There must be no smoking or eating (including chewing gum) in the laboratory. Keep
pencils, fingers etc out of your mouth.

5. Report all accidents to the lecturer or lab technician. Cuts on the hand must be
covered – see the lecturer or lab technician for a plaster.

6. Treat any spillages of culture or broken culture tubes immediately with disinfection
and report the spillage at once. Do not attempt to pick up pieces of broken glass,
report the breakage at once!
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ASEPTIC TECHNIQUE

In all microbiological work it is essential that aseptic precautions are taken in order to prevent
contamination of the culture in use, the operator and his/her environment and so that the
results of the experiments will not become meaningless because of contamination of media
by unwanted microorganisms.

It is necessary therefore that the microbiologist should develop a good aseptic technique and
practice it rigorously in the laboratory. The methods of handling cultures and apparatus will
be demonstrated during the practical classes and should be adhered strictly.

Aseptic techniques are used to prevent contamination of the microbiologist and the
laboratory. This is of utmost importance when pathogenic (disease producing) micro-
organisms are being handled. Since it is not always possible to recognize a pathogen without
a series of diagnostic tests, you must work on the assumption that all micro-organisms are
potentially pathogenic. If a culture is spilled, report it at once.
BIO461 MICROBIOLOGY

THE USE AND CARE OF THE MICROSCOPE

The compound microscope is a precision instrument composed of a series of lenses designed

and arranged to provide, under optimal conditions, maximum magnifications ranging form
approximately 1 – 2000 times (x) the diameter of the specimen being observed.

The ordinary compound microscopic consists of a series of optical lenses, mechanical

adjustment parts and supportive structures from various components. The optical lenses
include the ocular or eyepiece, usually three objectives with different magnifying powers, and
the substage condenser. The coarse, fine and condenser adjustments knob together with the
iris diaphragm level comprise the major mechanical parts concerned with the operation of the
instrument. The various components of the scope are held in position by or contain within,
supportive structures such as the base, arm, pillar, body tube(barrel), and revolving
nosepiece.

Successful operation of the microscope depends upon proper maintenance and correct use.
Here are some suggestions :

1. When carrying the instrument, grasp the arm with one hand, and place the other hand
under the base as a support. Keep the instrument in an upright position. In the event
the light source is not attached, take the scope to your laboratory table first, and return
for the light source. Do not carry both items at the same time.

2. Keep the microscope at least six inches from the edge of your laboratory table.

3. Do not handle the lenses with your fingers. Perspiration contains fatty acids and other
substances which can mar the lens glass. Always use the lens paper for the cleaning of
the optical system.

4. Wipe the lenses of the microscope before and after class use.

5. Do not temper with any components of the instrument. If the microscope does not
seem to be functioning, notify the instructor immediately. As other laboratory sections
may use the same instruments, any part previously damaged and subsequently
reported may be charged to you.

6. Do not allow chemicals to come in contact with any part of the instrument.

7. Remove immersion oil from the microscope with lens paper.

8. Always be certain that the low-power objective is in the working position, i.e. in line
with the body tube, before putting the microscope away.
BIO461 MICROBIOLOGY

PRACTICAL 1

A. EXAMINATION OF STAINED CELLS

The numbers and types of organisms seen under the microscope are limited by the
magnification of the objectives used. Because of their extreme small size bacteria are not
generally studied with the low or high dry objectives but instead they are stained with the oil
immersion objective. This practical will give you the experience in the use of oil immersion
and also serves as a comparative study of the stained cells between bacterial cells and typical
plant and animal cells.

Materials

2. Staphylococcus aureus
3. Bacillus subtilis
4. Salmonella typhimurium

Procedure

1. Examine all the slides under the oil immersion objectives.

2. Observe the size and shape and any visible structures of these organisms.

3. Make illustrations/drawings of all observations.

B. EXAMINATION OF LIVING BACTERIA

From the stained preparation of organisms used in Practical 2, you might believe that bacteria
are immobile, inanimate objects. They appear that way because the organisms are killed and
fixed when the slides were prepared. Unstained bacteria look somewhat different: many
species of bacteria are highly motile.

Direct examinations of living microorganisms can be extremely useful in determining the size
and shape relationships, motility, and reactions to various chemicals or immune sera. Two
methods are generally used:
Hanging drop and wet mount techniques.

Both methods maintain the natural shape of organisms and reduce the distorted effects which
can occur when specimens are dried and fixed.

In this practical, the living motile and non-motile bacteria will be observed. Non-motile
bacteria will exhibit Brownian movement, in which the cell bounces about aimlessly for short
distance. Motile organisms usually move for considerable distance in a particular direction,
though sometimes spinning or rolling is seen.
BIO461 MICROBIOLOGY

Materials.

1. 24-hour nutrient broth cultures of

(a) Staphylococcus aureus
(b) E. coli

2. Hollow ground depression slides

3. Vaseline

4. Inoculating loops

5. Glass slides and cover slips

6. Applicator sticks

I. HANGING DROP TECHNIQUE

This technique is used to determine whether living bacteria are flagellated and hence motile.
This can be of diagnostic importance when followed by the flagellum stain. Care should be
taken since LIVING BACTERIA are used. Remember to treat all microbial cultures as if they
were pathogenic.

Procedure

1. With the aid of a wooden applicator stick apply Vaseline to the edges on one surface
of the cover slip and place it on the lab table so that the Vaseline treated surface faces
upward.

2. Place on the center of the cover slip a drop of bacterial broth culture using a sterile
inoculating loop.

3. Invert a depression slide and lower it onto the prepared cover slip. Gently press the
slide down so that the Vaseline forms a seal between the slide and the coverslip.

4. Carefully turn the slide over, making sure that the drop does not run sideways.

5. Focus on the drop using the x10 objective. Change to the x40 objective and reduce the
light intensity.

6. Observe the type of movement:

a. Drifting of the bacteria across the field of view caused by evaporation of the
drop through an incomplete Vaseline seal.
b. Brownian motion.
c. True motility-a characteristic of flagellated bacteria. The bacteria will be
observed darting about and changing direction.

7. Record your findings.

BIO461 MICROBIOLOGY

NOTE

1. Make one slide at a time and examine quickly since there will be limited supply of
oxygen around the drop.

2. After examination, place the slide preparations in the jars of disinfectant provided.

II. TEMPORARY WET MOUNT TECHNIQUE

Procedure

1. Flame the inoculating loop to redness.

2. Remove the plug from the bacterial culture to be used and flame the mouth of the
tube.

3. Remove a loopful of the culture and place it in the center of a clean glass slide.

4. Flame the mouth of the tube and return the plug.

5. Sterilize the inoculating loop.

6. Put a cover slip over the bacterial suspension and press down gently.

7. Examine the preparation under low power and then under high power objectives.
BIO461 MICROBIOLOGY

C. THE STAINING OF MICROORGANISMS

Bacterial morphology may be examined in two ways:

1. By observing the living, unstained organisms as is done in demonstrating bacterial

motility

2. By observing dead cells stained with dyes.

Living bacteria are almost colorless and do not present sufficient contrast with the water in
which they are suspended to be clearly visible. Staining the organisms will make them
contrast in color with their surroundings, so they are more readily visible. Certain stains can
also be used to identify certain internal structures of the cell, which would otherwise pass
unseen. Further, in order to use the oil immersion objective and obtain the greatest degree of
magnification, it is more convenient to use stained preparations than wet mounts.

PREPARATION OF FILMS FOR STAINING

I. From broth cultures

1. Sterilize an inoculating loop, allow it to cool and remove one drop of bacterial
suspension from the tube.

2. Place the drop in the center of a clean, grease free microscope slide and spread the
drop out to form a thin film towards the top end of the slide. Do not go too near the
edges of the slide.

3. Allow the film to dry by wafting the slide gently above a Bunsen flame.

4. Pass the dried smear, film uppermost, quickly once or twice through the Bunsen
flame. CARE !.
This will fix the bacteria i.e. kill them and stick them firmly to the slide.

5. Place the slide on a staining rack and apply the stains.

II. From agar cultures

1. Place a drop of sterile water in the center of a slide.

2. Sterilize the loop and remove a minute quantity of the bacterial culture.
DO NOT scrape the loop across the culture but simply touch the loop to the culture.

3. Mix the cells in the drop of water and proceed as in the case of broth cultures. (Steps
2-5)
BIO461 MICROBIOLOGY

DIRECT STAINING WITH BASIC DYES

Dyes are generally salts in which one of the ions is colored. A salt is a compound composed
of a positively charged ion and a negatively charged ion. For e.g. methylene blue is actually
the salt methylene blue chloride which dissociates as follows:

MBC ---> Methylene blue + chloride

The color of the stain is the positively charged methylene blue ion. Bacterial cells have a
slight negative charge when the pH is near neutrality, whereas the surroundings are generally
neutral. This negatively charged cell combined with the positively charged methylene blue
ion, with the results that the cells appear stained.

Dyes may be divided into two groups – basic or acidic. If the color is in the positive ions, we
call it a basic dye. If the color is in the negatively charged ion of the dye, we call it an acidic
stain.

In this practical, basic dyes methylene blue, crystal violet and carbol fuchsin will be used.
Methylene blue reacts with the negatively charged cell at the slowest rate taking 30-60
seconds to stain the smear properly.
Crystal violet is more reactive and requires only 10 seconds to stain a microbial preparation.
Carbol fuchsin is an even more powerful dye, requiring only 5 seconds.
CARE! Do not overstain.

SIMPLE STAINING TECHNIQUES

Materials

1. 24 hours broth cultures of

a. E. coli
b. Staph. aureus

2. 24 hours nutrient agar slants of

a. Bacillus subtilis
b. Pseudomonas aeruginosa

3. Slides

4. Inoculating loop

5. Dye solutions
a. crystal violet
b. methylene blue
c. carbol fuchsin

6. Test tubes
Procedure

1. Collect some saliva in the test tube provided.

2. Prepare the film for staining.

3. Flood the film with approximately 5 drops of the stain and allow time for the stains to
react:
Methylene blue 30 seconds, crystal violet 10 seconds, carbol fuchsin 5 seconds.

4. Wash off the stain with tap water, blot off excess water and dry the stained film high
over a Bunsen flame.

5. Examine the slide directly i.e. without the cover slip under the oil immersion
objective lens (x100)

6. Draw labeled diagrams to show the morphological shapes of the bacterial cells you
have just examined.
Always remember to include the magnification to give scale to your diagrams.

7. Record your results

NOTE : For oil immersion examination, the slides MUST BE DRY

0
BSc (Hons.) Biology - BIO 461

PRACTICAL 2

A. DIFFERENTIAL STAINING - THE GRAM STAIN

Differential stains are so named because they do not stain all kinds of bacteria equally.
Microorganisms differ from one another chemically and physically and thus may react
differently to the given stains. For example, the Gram stain differentiates between the two
cell wall types in prokaryotic cells. The Spore stain, stain the spore and the vegetative cell in
different colors.

The Gram staining technique is the most widely used differential staining technique in
bacteriology, and was first developed by Christian Gram in the 1880’s. It is used primarily to
divide bacteria into two broad groupings:

Gram-positive and Gram-negative cells.

This procedure is of considerable importance in bacterial taxonomy, and it also indicates

fundamental differences in cell wall structures in bacteria.

The Gram stain requires four different solutions: a basic dye, a mordent, a decolorizing agent
and a counterstain.

Cells are first stained with crystal violet, a basic dye, followed by iodine which forms a
complex inside the cells. A mordent is a substance which increases the affinity or attraction
between the cell and the dye. Examples of mordent are acids, bases, metallic salts and iodine.
Under the action of a mordent, a cell is more strongly stained, and it is more difficult to wash
out the stain after the application of the mordent.

Decolorization is then performed with alcohol. A decolorization agent is a substance which

removes the dye from a stained cell. The cells which retain the basic dye after decolorization
are called Gram positive and those that lose the stain are called Gram negative. After
decolorization, a counterstain safranine is used to make visible the now decolorized Gram
negative bacteria.

Gram-positive cells stain blue/purple

Gram-negative cells stain red/pink

BSc (Hons.) Biology - BIO 461

Materials

24 hours nutrient broth cultures of

a) E. coli
b) Staphylococcus aureus
c) Bacillus subtilis

1. Slides

2. Gram stain reagent sets

3. Immersion oil

Procedure

1. Prepare a fixed smear of the bacterial cells.

2. Flood the smear with crystal violet for one minute.

3. Pour off the stain and holding the slide at an angle, pour on the iodine solution so that
it washes away the crystal violet. Cover the slide with fresh iodine and allow it to act
for 2 minutes.

4. Rinse the slide in slow running tap water.

5. Treat the smear with alcohol. The alcohol must not be allowed to remain on the slide
but must be dripped on and allowed to drain off the slide until the color ceases to
come out of the preparation.

6. Immediately wash the slide with tap water.

7. Counterstain with safranin for 30 seconds.

8. Rinse as above and blot dry with blotting paper.

9. Examine directly under oil immersion.

10. Record your observations.

COMMENTS

Certain species of bacteria do not give a consistent reaction to the Gram stain - sometimes
reacting positively and sometimes negatively. These bacteria are said to be Gram variable.
Older or dead Gram-positive cell sometimes lose their Gram positiveness and may also
appear Gram-negative. The best Gram stains are therefore given by fresh cultures.
BSc (Hons.) Biology - BIO 461

B. STRUCTURAL STAINING

I. THE ENDOSPORE STAIN

Many organisms other than bacteria form spores, but the bacterial endospore is unique in its
degree of resistance to heat, drying, radiation, chemicals, and disinfectants.

Spore formation is primarily limited to two genera: Bacillus and Clostridium. The Bacillus
spp. is aerobic while the Clostridium spp. is anaerobic.

This exercise will demonstrate the Schaeffer-Fulton technique for the differentiation of
spores and vegetative cells. In this procedure, an aqueous primary dye is applied to a
specimen and steamed to enhance penetration of the relatively impermeable spore coat.

The endospores will be stained green

The vegetative cells will be stained red.

Materials

1. 48-hour nutrient agar slant cultures of Bacillus subtilis.

2. Schaeffer-Fulton staining set :

a. Malachite green
b. Safranin

3. Slides

Procedure

1. Prepare a fixed smear of the culture provided.

2. Flood the smear with 5% aqueous malachite green and heat to steaming for 1-2
minutes.

3. Allow the slide to cool. DO NOT allow the stain to dry out at this stage.

4. Rinse carefully under running water.

5. Flood with safranin for 1 minute.

6. Rinse very lightly in water, blot dry.

7. Examine under oil immersion objective.

8. Record all your observations.

BSc (Hons.) Biology - BIO 461

COMMENTS

The safranin counterstain replaces the malachite green in the vegetative cells by the process
of dye exchange. It does not replace the green dye in the endospore because it cannot
penetrate this structure. It is also easily washed out of the cells therefore washing/rinsing
should be kept to a minimum in step 6 above.

II. THE CAPSULE STAIN.

Many bacteria synthesize loose amorphous organic polymers which are deposited outside the
cell wall as capsules or slime layers. A capsule refers to the layer tightly attached to the cell
wall, whilst a slime layer is the loose structure that often diffuses into the medium.

Primary interest in these exopolymers arise from determining their role in pathogenicity
(disease production) since many pathogenic bacteria are capsulated. Capsules appear to
increase the virulence of organisms by protecting them from the defense mechanism of the
host.

The composition of the polymers varies from species to species e.g. in Acetobacter xylinum
the capsule is composed of cellulose (β– glucoseⁿ), whilst in Leuconostoc it may be either
Levan (fructoseⁿ) or Dextran (glucoseⁿ).

Other examples of bacteria that produce capsules are Diplococcus pneumoniae, Clostridium
perfringens and Klebsiella pneumoniae. Streptococcus, Acetobacter, Rhizobium, Bacillus are
also among many genera that produces them.

Materials

1. 24 hours nutrient broth culture of Klebsiella aerogenes

2. Indian ink

3. Safranin

4. Glass slide

Procedure

1. Place several loopfuls of the culture onto a clean glass slide near the edge.

2. Place an equal amount of Indian ink or nigrosin near the culture.

3. Mix well with the loop. Then spread the mixture along the slide using the blood smear
technique which the instructor will demonstrate.
BSc (Hons.) Biology - BIO 461

CARE ! Aseptic precautions must be observed here since living bacteria are involved.

4. Allow to dry.

5. Cover the smear with safranin for 10 seconds, rinse carefully and blot dry.

6. Examine the slide using the x40 objective and adjust the light to improve the contrast.
BSc (Hons.) Biology - BIO 461

PRACTICAL 3

A. THE PURIFICATION OF BACTERIAL CULTURES – PURE CULTURE

TECHNIQUES

It is essential that pure cultures be utilized in order to identify and characterize bacteria. A
pure culture is one in which all cells are descendants of a single cell.

Bacteria can be separated from each other in a mixture of bacteria by progressively diluting
the mixture until single bacteria cells are so widely spaced that they will grow, divide and
produce separate colonies. Two dilution methods are generally used to achieve this aim:

1. Streak plate method

2. Pour plate method.

I. STREAK PLATE METHOD

Materials

1. Nutrient broth culture of E. coli

2. Nutrient broth culture of Serratia marcescens

3. Nutrient agar plates

4. Tube containing 10 ml of sterile saline

5. 4 tubes containing 12-15 ml of nutrient agar (melted and in the water bath at 45˚C-
50˚C)

6. Sterile Petri dishes

Procedure

1. Prepare 4 plates of Nutrient Agar (NA).

2. Mark the plates on the underside into 4 sectors A, B, C, D.

3. Sterilize an inoculating loop and transfer a drop of E. coli to sector A. Draw the loop
over the surface of the agar (DO NOT CUT INTO THE AGAR) in a series of parallel
streaks.

4. Re-sterilize the loop, allow it to cool and draw a series of parallel streaks in sector B
cutting across the ends of the first streak in sector A.

5. Re-sterilize the loop, allow it to cool and draw a series of parallel streaks in sector C
cutting across the end of the streaks in sector B.
BSc (Hons.) Biology - BIO 461

6. Re-sterilize the loop, allow it to cool and draw a series of parallel streaks in sector D
cutting across the streaks in sector C BUT NOT touching the original streaks in sector
A.

7. Do the same with Serratia marcescens culture.

8. After streaking the plates above, carefully pour the culture of E. coli into the culture
of Serratia marcescens to obtain a mixed suspension.

9. Prepare a streak plate of the mixed culture following the procedures above.

10. Incubate all plates at 37˚C for 2 days and then at 30˚C to allow Serratia marcescens to
develop its characteristic red pigment.

11. After 2 days, examine the cultures which you have inoculated and evaluate your
streaking effectiveness in producing pure colonies.

II. POUR PLATE METHOD

Each student will prepare the pour plates with the mixed culture of E. coli and Serratia
marcescens.

Procedure

1. Transfer one loopful of the mixed culture to the tube of sterile saline. Mix the tube
well by rotating the tube between the hands.

2. Transfer two loopfuls from the saline to a tube of melted agar (label it as tube 1).

3. Mix thoroughly and transfer two loopfuls from tube 1 to a second tube of melted agar
(label it as 2). Quickly pour the contents of tube 1 into an identically labeled Petri
dish.

4. Repeat the procedure for tubes 2 and 3.

5. Do not inoculate the fourth tube but pour the agar from this tube into a Petri dish as a
control.

6. Invert the Petri dishes after the agar hardens.

7. Incubate all plates and tubes at room temperature.

The following day

1. Examine the cultures and evaluate your plate techniques in producing pure colonies.

2. Compare the appearance of sub-surface and surface colonies on the pour plates.
BSc (Hons.) Biology - BIO 461

PRACTICAL 4

A. CULTURE MEDIA PREPARATION

In order to support the growth of microorganisms in the laboratory, it is necessary to use

culture media containing all the essential nutrients for microbial growth. Most heterotrophic
organisms require a source of energy (carbon source), a source of nitrogen, a source of
accessory inorganic elements and trace elements, accessory growth factors (usually organic
e.g. vitamins, amino acid), and water. However, the nutritional requirements of individual
bacteria are extremely variable ranging from organisms which can utilize very simple
inorganic compounds and elements to those which require complex substances. Many
bacteria which are pathogenic for animals and man require relatively complex media and are
said to have “fastidious” nutritional requirements.

A wide variety of dehydrated bacteriological media is available commercially. The selection

of an appropriate culture medium for a specific procedure is normally made by referring to
suitable texts and commercial manuals.

Simple or synthetic medium

This type of medium is formulated from a mixture of chemically definable nutrients and can
be used for those organisms having simple nutritional requirements and not requiring
complex nutrients.

Complex or natural medium

This consists of mixtures of animal and/or plant extracts to which various organic compounds
are added, although they contain all the necessary factors for growth. They are used when the
nutritional requirements of a microorganism are not known or when the microorganism is
unable to grow without the complex growth factors provided by the medium.

Nutrient broth

Nutrient broth (a solution of beef extract and peptone) is the basis of most media used for the
cultivation of most common pathogenic and nonpathogenic bacteria. Another type of nutrient
broth commonly used in bacteriology is the infusion broth. Infusion broths contain meat
infusion (prepared by extracting ground meat with water at low temperatures). Peptones are
inorganic salts. A wide variety of infusion broths are available commercially including those
prepared from beef, veal, heart, liver and brain. Nutrient broths prepared with meat extracts
are generally less expensive than infusion broths.

Solid media

Solid media may be prepared from nutrient broth by the addition of agar or gelatin. Agar,
which is used most often as a solidification agent is prepared from a variety of seaweed. The
advantages of agar as a solidification agent are its property of remaining as agent at various
incubation temperatures and the fact that it is not metabolized by most bacteria.
BSc (Hons.) Biology - BIO 461

Keeping in mind the concept that most media consist of a nutrient broth base to which agar is
added when a solid medium is required, a variety of media may be prepared by adding
substances to the basic materials.

Enriched media : is prepared by the addition of inhibitory substances such as dyes, bile salts
or sodium chloride in various concentrations.

Selective media : allow some organism to grow while retarding the growth of others and is
particularly valuable in isolating certain bacteria from materials such as feces which contain
more than one organism.

Differential media : is prepared by adding various metabolites such as carbohydrates which

may be acted upon in different ways by different bacteria.

Complex media : may be enriched and differential, selective and differential or enriched,
selective and differential. For e.g. Blood agar containing crystal violet and sodium azide is
enriched due to the addition of blood, is selective due to the inhibitory effects of crystal violet
and sodium azide and is differential since hemolytic reactions may be observed.

Examples of laboratory media.

1. Simple medium : Czapek-Dox Agar for Fungi

Sucrose : 3%
K2HPO4 : 0.1%
NaNO3 : 0.3%
MgSO4.7HO : 0.05%
KCL : 0.05%
Fe SO4 : 0.001%
Agar : 1.5%
In distilled water

2. Complex medium : Nutrient Agar for Bacteria

Lab Lemco(meat extract) : 0.1%

Peptone(hydrolysed protein) : 0.5%
Yeast extract : 0.2%
NaCl : 0.5%
Agar : 1.5%
In distilled water
BSc (Hons.) Biology - BIO 461

Materials

1. 500 ml Erlenmeyer flasks

2. Tripod and wire gauze

3. Sterile Petri dish

4. Sterile 10ml pipettes

5. Standard culture tubes

6. 1 tube containing 5ml sterile defibrinated bovine blood

7. 250ml conical flask containing 100ml sterile nutrient agar

8 Dehydrated nutrient agar

9. Dehydrated nutrient broth

Procedure

I. Solid Media

Prepare 5 agar deeps, 5 agar slants, and 4 blood agar plates according to the following
procedure:

7. Suspend the required amount of dehydrated nutrient agar in the required amount of
tap water.

8. Heat the suspension to boiling, to completely dissolved the dehydrated powder.

NB : The solution will be clear when the agar is completely dissolved.

CARE! Do not heat the agar too rapidly. When the medium approaches the boiling point,
lower the flame to prevent excessive foaming.

9. Agar deeps – prepare 5 agar deeps by pipetting 10ml of the melted nutrient agar.

10. Agar slants – prepare 5 agar slants by pipetting 5ml of melted nutrient agar into each
of the 5 standard culture tubes.

11. Place the deeps and the slants in the rack to be autoclaved at 15lbs-pressure for 15
minutes at 121˚C.

12. After sterilization, the deeps will be allowed to solidify in the upright position and the
slants placed at an angle so that they solidify with about ½ inch butt.
BSc (Hons.) Biology - BIO 461

13. Blood agar plates – using aseptic technique, add 5ml of sterile defibrinated blood to
the 100ml of sterile nutrient agar which has been allowed to cool to 50˚C.
Immediately pour 4 blood agar plates.

CARE !

14. When adding the blood to the melted agar, the blood should be allowed to flow down
the side of the flask while the flask is gently rotated. Vigorous shaking will cause the
formation of bubbles which will result in plates with a pitted surface.

15. If the media is too hot, the blood will turn brown and the so called “chocolate agar”
will result. If the media is too cold, the agar will begin to solidify and the plates will
have an irregular surface. The temperature of the blood should be close to room
temperature when it is added to the media.

16. Allow your plates to solidify and place them media-side up.

NOTE :

Agar plates are always stored and incubated in an inverted position to prevent the water
of condensation from dripping onto the agar surface. An excessive moist agar surface
makes the isolation of individual colonies difficult.

17. Expose 2 blood agar plates for 5 minutes to the environment and label them E.
Incubate the 4 blood plates at 37˚C. During the next lab period examine the blood
plates for growth.

II Fluid Media

Prepare the required amount of dehydrated nutrient broth in the required amount of water.

Procedure

1. Suspend the required amount of dehydrated nutrient broth in the required amount of
water.

2. Heat to dissolve the powder completely.

3. Pipette 6ml of nutrient broth into each of the tubes.

4. This media will be autoclaved along with the solid media.

NOTE: Omit the sterilization of a few tubes and plates of each type of media to
emphasize the necessity of sterilization.
BSc (Hons.) Biology - BIO 461

III. The following day

1. Compare the media which was sterilized with that which was not sterilized.

2. Record all your observations.

B. EXAMINATION OF BACTERIAL PLATES

Bacterial species can sometimes be identified on the basis of how they appear on or in the
different media. The pigmentation, size and shape of bacterial colonies as they grow on and
in agar plates can provide identifying signs. Observing the growth characteristics of
organisms in broth cultures can also be helpful.

The major problems that may arise are contaminations and species variations. These must be
eliminated if good results are to be obtained. Species variations can be the results of:
i. An environmentally induced modification of a genetically controlled characteristic.
ii. Spontaneous undirected mutation.
iii. The acquiring of a new assortment of genes as a consequence of sexual reproduction.

The first factor listed above generally is temporary in its effect i.e. it manifest itself only as
long as the environment conditions are present. The second and third factors exert a
permanent influence.

Materials

All the materials listed below have been prepared beforehand.

2. Nutrient agar plates of:

a. Sarcina lutea and Serratia marcescens (25˚C)

b. Bacillus subtilis and E.coli (37˚C)
c. Streptococcus fecalis and Pseudomonas fluorescens (25˚C)

3. 24 hours pour plate preparation of:

b. Serratia marcescens (37˚C)

c. Sarcena lutea (37˚C)
d. Bacillus subtilis (37˚C)
e. E.coli (37˚C)

4. 24 hours agar slant of the following cultures incubated at 25˚C:

a. Bacillus subtilis
b. E.coli
c. Serratia marcescens
d. Pseudomonas fluorescens

5. 48 hours nutrient broth culture of the following incubated at 25˚C:

BSc (Hons.) Biology - BIO 461

a. Bacillus subtilis
b. Serratis marcescens
c. E.coli

Procedure

1. Examine the various cultures provided and record all your observations.

2. Be careful not to disturb/shake the broth preparations as this may dislodge the surface
growth.

3. All results should include the following observations.

b. colony description
- estimated diameter
- whole colony appearance
- margin characteristic
- elevation properties
- pigmentation if any

c. broth characteristics: turbidity, presence of flocculent, presence of sediment,

presence of pellicle, color

d. agar stroke properties/patterns of growth on agar slants: branched, beaded, even,

rhizoid, spreading

4. Also comment upon the number and size of the colonies

CARE! – Aseptic technique is important here because of the potential pathogenicity of

your isolates : TRY TO KEEP THE LIDS ON YOUR PETRI DISHES
DURING YOUR EXAMINATIONS.
BSc (Hons.) Biology - BIO 461

PRACTICAL 5

BIOCHEMICAL ACTIONS OF BACTERIA

Individual and colonial morphology is seldom sufficiently distinctive to characterize bacteria.

Therefore, physiological and immunological properties are utilized. Bacteria differ in their
abilities to metabolize various substrates and in the end products formed.

A. CARBOHYDRATE METABOLISM

Bacteria utilize a wide range of carbohydrates both oxidatively (aerobically) and

fermentatively (anaerobically).

1. Fermentation of sugars

Materials

1. Glucose broths with Durham tubes and phenol red indicator

2. Lactose broth with Durham tubes and phenol red indicator

3. Sucrose broths with Durham tubes and phenol red indicator

4. 18 hour nutrient broth cultures of E.coli and Salmonella typhimurium

Procedure

1. Inoculate the small bottles containing the different sugars at a 1% concentration with
a loopful of the organisms provided. The bottles contain an acid indicator (e.g. phenol
red: red -→ yellow under acidic conditions) and a small inverted Durhams tube to
collect any gas which may be produced (usually CO2 and H2).

2. Incubate them at 37˚C.

3. Record your observations as acid and/or gas production (e.g A+G+, A+G- etc.).
BSc (Hons.) Biology - BIO 461

2. Hydrolysis of starch

The ability of some microorganisms to hydrolyse starch is useful for identification purposes.
Iodine reacts with starch to form a blue-black complex, with dextrin it forms a red colored
complex.

Materials

1. Starch agar plates

2. Broth cultures of Bacillus subtilis and E.coli

Procedure

1. Streak a starch agar plate with each of the bacteria provided which will result in
isolated colonies as in Practical 9.

2. Incubate the plates at 37˚C.

The following day

1. After incubation, test for starch hydrolysis by flooding the plates with Gram’s iodine.

2. Examine the plates and note the colonies that show clear uncolored zones in contrast
with the blue-black background of the starch-iodine complex.

3. Indicate the extent of the zones of hydrolysis and whether or not reddish colored
zones are seen.

4. Record all your results and observations.

B. PROTEINS AND AMINO ACIDS METABOLISM

1. Indole test

Some organisms produce indole as an end product of tryptophan utilization.

Materials

1. Broth cultures of Bacillus subtilis, E.coli, Salmonella typhimurium

2. 3 tubes of tryptone broth

3. Kovac’s indol test reagent

BSc (Hons.) Biology - BIO 461

Procedure

1. Inoculate tubes of peptone water with a loopful of the test organism.

2. Incubate the tubes.

The following day

1. After incubation add a few drops of Kovac’s indole reagent

(p-dimethylaminobenzaldehyde).

2. A red/dark color indicates the presence of indole.

2. Hydrogen sulphide

Materials

1. 3 Kigler’s slant

2. Broth culture as above

Procedure

1. Inoculate the Kigler’s slant by the stab method.

2. Incubate.

The following day

1. Observe the Kigler’s slant for the production of H2S. The presence of black precipitate
along the line of growth in the Kigler’s slant is evidence that H2S has been produced.

2. Record all your observation.

3. Gelatin hydrolysis test

This test indicates proteolytic activity.

Materials

1. Gelatine agar plates

2. Broth cultures as above

BSc (Hons.) Biology - BIO 461

Procedure

1. Inoculate the plates of gelatin agar (NA+4% gelatin) with a single central streak.

2. Incubate.

The following day

1. Flood the plates with mercuric chloride solution. CARE!

2. The medium becomes opaque in regions still containing gelatin and clear in regions
where gelatin has been hydrolysed.

C. VOGES-PROSKAUER TEST

Some bacteria ferment glucose and produce acetoin as an intermediate compound in 2,3
butanediol formation.

Materials

1. 2 tubes of Clark-Lub’s medium (MR-VP medium)

2. Broth culture of E.coli, Klebsiella spp.

Procedure

1. Inoculate the cultures into the Clark-Lubs medium.

2. Incubate overnight at 37˚C.

The following day

1. Perform the Voges-Proskauer test on the Clark-Lubs broth culture.

2. Add 0.5ml of KOH-creatine solution.

3. Shake the tube vigorously for 30 seconds OR

4. Mix 2ml culture fluid with 5ml of 40% KOH and add a trace of creatine. Shake and
allow to stand for up to 60 minutes.

5. A red/pink color indicates the presence of acetoin.

BSc (Hons.) Biology - BIO 461

D. CATALASE TEST

Catalase is an iron-porphyrin containing enzyme that decomposes hydrogen peroxide to

water and oxygen molecule.

Materials

1. Nutrient agar slants

2. Broth culture of Streptococcus spp. Staphylococcus spp.

Procedure

1. Inoculate the nutrient agar slant with the cultures.

2. Incubate at 37˚C.

The following day

1. Perform the catalase test by adding several drops of a 5% solution of hydrogen

peroxide.

2. A positive test is indicated by the rapid evolution of oxygen with vigorous bubbling.

E. UREASE TEST

Some bacteria break down urea to produce ammonia and carbon dioxide.

Materials.

1. 5 urea broth with indicator

2. Nutrient broth cultures of E. coli, P. vulgaris, S. marcescens, Pseudomonas

fluorescens

Procedure

1. Inoculate the medium (contain peptone, glucose, urea, phenol red indicator) with a
loopful of the test organism.

2. Incubate overnight.

The following day

1. After incubation urease-positive organisms produce an intense red/purple coloration

of the medium.
BSc (Hons.) Biology - BIO 461

F. NITRATE REDUCTION TEST

Many microorganisms can reduce nitrate in a number of different reactions. Some bacteria
reduce nitrates to nitrites while others reduce nitrates completely to ammonia.

Materials

1. 5 tubes of nitrate broth (containing 0.1% KNO3)

2. Nitrate test reagent

3. Nutrient broth cultures of E. coli, Proteus vulgaris, Serratia marcescens,

Pseudomonas fluorescens

Procedure

1. Inoculate the cultures into the nitrate broth.

2. Incubate overnight.

The following day

1. After incubation test a 1ml sample of the culture medium with 1ml of Follet and
Ratcliff’s reagent. (F & R reagent)

2. An orange/brown color indicates the presence of nitrite.

3. An absence of nitrite indicates that:

a. there has been no nitrate reduction
b. the reduction has proceeded beyond the nitrite stage

4. In the absence of an orange/brown color (in the tube above), add a small amount of
powdered cadmium to the tube. If nitrate is still present, it will be catalytically
changed to nitrite which will then react with the F & R reagent in the tube.

5. In the absence of a positive nitrite result, look for bubbles of N2 gas in the Durhams
tube OR

6. Test a fresh 1ml sample of the culture fluid with 1ml of Nesslers reagent. An
orange/brown color indicates the presence of ammonia.
BSc (Hons.) Biology - BIO 461

PRACTICAL 6

ANTISEPTIC EVALUATION BY THE FILTER PAPER DISC METHOD

Introduction

The term antiseptic has unfortunately been somewhat ill defined. Originally the term was
applied to any agent that prevents sepsis or putrefaction. Since sepsis is caused by growing
microorganisms, it follows then that an antiseptic inhibits microbial multiplication without
necessary killing them. By this definition we can assume that antiseptics are essentially
bacteriostatic agents.

If we are to compare antiseptics on the basis of their bacteriostatic properties, the filter paper
disc method is a simple and satisfactory method to use. In this method a disc of filter paper
(half inch diameter) is impregnated with the chemical agent (antiseptic) and is placed on a
seeded nutrient agar plate. The plate is incubated for 48 hours. If the substance is inhibitory, a
clear zone of inhibition will surround the disc. The size of this zone is an expression of the
agent’s effectiveness and can be compared quantitatively against other substances. In this
experiment, we will measure the relative effectiveness of three agents against two organisms:
Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative).

Materials

1. Chemical agents to be tested

2. Cultures of S. aureus, P. aeruginosa and E. coli

3. Sterile Petri dishes

4. Liquid nutrient agar

5. Sterile filter paper disc

Procedure

1) Label the bottom of a Petri dish with the name of the organisms, the chemical agent and
your name and date.

2) Take one or two loopfuls of your test organism from the cultures provided and aseptically
inoculate a tube of liquid nutrient agar at 50˚C. Mix carefully and pour into a sterile Petri
dish.

3) After the medium has solidified in the plate, pick up a sterile filter paper disc with flamed
forceps, dip the disc halfway into a beaker of the chemical agent and carefully place the
disc in the center of the plate. To secure disc to the plate, press lightly with forceps.

4) Incubate the plates for 48 hours at 37˚C.

The following day

BSc (Hons.) Biology - BIO 461

1. Measure the zone of inhibition from the edge of the disc to the edge of the growth. It
should be measured on the bottom of the plate, not on the surface of the medium.

2. Fill in the master chart in the laboratory with your results FOR EACH GROUP so that
you would obtain replicated results.

Chemical agent S. aureus P. aeruginosa E. coli

5% phenol

5% formaldehyde

5% iodine
BSc (Hons.) Biology - BIO 461

PRACTICAL 7

BACTERIAL AMYLASE

Introduction

Enzymes are proteins produced by many animals, plants and microorganisms and are used
widely in the laboratory, industry and in the medical field. With the advance of industrial
microbiology, enzymes from microbes have become a very important source. The main
organisms used in the production of enzymes are shown below:

Table 1: ENZYMES FROM MICROORGANISMS

ORGANISMS ENZYME
FUNGI

Aspergillus oryzae Amylase, protease

Aspergillus niger Amylase, glucosidase, cellulase,

pectinase, glucose oxidase, catalase

Rhizopus spp. Amylase, pectinase, lipase

Mucor pusillus Rennin

BACTERIA

Bacillus subtilis Amylase, protein, alkaline protease

Micrococcus lysodeikticus Catalase

Pseudomonas myxogenes Rennin

YEAST

Saccharomyces cerevisiae Invertase

Saccharomyces fragilis Lactase

In this practical, students will learn how to:

1) isolate amylase producing bacteria

2) demonstrate amylase activity

BSc (Hons.) Biology - BIO 461

A. ISOLATION OF AMYLASE PRODUCING BACTERIAL STRAINS FROM THE

SOIL

Most organisms from the genus Bacillus show high amylase activity. This genus usually
forms heat resistant spores and heat shock treatment may be used to kill off all vegetative
cells from a soil sample, leaving only Bacillus and Clostridium spread on starch agar and
incubated at room temperature under aerobic conditions. Only the Bacillus spores would then
germinate as Clostridium is anaerobic. Amylolytic colonies may then be demonstrated by the
reaction of iodine upon starch.

Materials.

1) Soil samples

2) Boiling tubes with cotton plugs

3) Tripod and wire gauze

4) 500ml beaker

5) Water baths set at 80˚C and 90˚C

6) Starch agar plates

7) Pasteur pipette

8) Alcohol

9) Bent glass rod (hockey stick) spreaders

10) Spatula

Procedure

1) Place a little soil in a boiling tube with a spatula.

2) Add about 50ml tap water and close with a cotton plug.

3) Shake the tube to break up particles of soil.

4) Perform heat shock treatment at 80˚C, 90˚C and 100˚C for periods of 10, 20 and 30
minutes.

5) Cool under running tap water.

6) Place a drop of heat shocked soil suspension on a starch agar plate with a sterile Pasteur
pipette and spread with a sterile bent glass rod. CARE ! Cool glass rod before spreading.

7) Perform this step with all nine boiling tubes.

BSc (Hons.) Biology - BIO 461

8) Incubate all plates at 30˚C for 48 hours.

9) Record the number of colonies on each plate as below:

COLONIES ON STARCH AGAR PLATES

TREATMENT TOTAL NUMBER AMYLASE POSITVE

80˚C/10min
80˚C/20min
80˚C/30min
90˚C/10min
90˚C/20min
90˚C/30min
100˚C/10min
100˚C/20min
100˚C/30min

B. DEMONSTRATION OF AMYLASE ACTIVITY

Amylase activity is indicated by a clear or yellow zone around colonies when the plates are
flooded with iodine. This is due to the fact that the starch has been broken down to simple
sugars that do not form the standard purplish starch/iodine complex. Thus this simple test
may be used to identify amylase producing strains.

Materials

1) iodine solution

2) slides

3) methylene blue

4) starch agar plates from practical A above

5) simple staining kit

BSc (Hons.) Biology - BIO 461

Procedure

1. Flood plates with iodine solution.

2. Pour off excess iodine.

3. Observe colonies with a zone of discoloration around them and record your result.

4. Pick up an amylase positive colony and perform simple staining.

5. Observe under oil immersion.

6. Record all your observations as such:

i. appearance of amylase positive colonies after iodine reactions.

ii. appearance of amylase positive organisms after staining.